Dhavarasa, P. | bioRxiv Science

bioRxiv2023

CRISPR Screening in Tandem with Targeted mtDNA Damage Reveals WRNIP1 Essentiality

A major impediment to the characterization of mtDNA repair mechanisms, in comparison to nuclear DNA repair mechanisms, is the difficulty of specifically addressing mitochondrial damage. Using a mitochondria-penetrating peptide, we can deliver DNA-damaging agents directly to mitochondria, bypassing the nuclear compartment. Here, we describe the use of a mtDNA-damaging agent in tandem with CRISPR/Cas9 screening for the genome-wide discovery of factors essential for mtDNA damage response. Using mitochondria-targeted doxorubicin (mtDox) we generate mtDNA double-strand breaks (mtDSBs) specifically in this organelle. Combined with an untargeted Dox screen, we identify genes with significantly greater essentiality during mitochondrial versus nuclear DNA damage. We characterize the essentially of our top hit - WRNIP1 - observed here for the first time to respond to mtDNA damage. We further investigate the mitochondrial role of WRNIP1 in innate immune signaling and nuclear genome maintenance, outlining a model that experimentally supports mitochondrial turnover in response to mtDSBs.

biochemistry↗

bioRxiv2020

NOX4 links metabolic regulation in pancreatic cancer to endoplasmic reticulum redox vulnerability and dependence on PRDX4

Pancreatic ductal adenocarcinoma (PDAC) is an almost universally fatal malignancy and there is an urgent need for new therapeutic targets. PDAC cells harbor genetic alterations and display metabolic changes that render them vulnerable to perturbations in redox homeostasis. Peroxiredoxin 4 (PRDX4) supports redox homeostasis by metabolizing H2O2 in the endoplasmic reticulum (ER). Based on functional genomics, we found that PDAC cell lines are dependent on PRDX4 for their growth and survival. We validated this dependency in established and primary PDAC cells, as well as 3D models and orthotopic xenografts. Cell death induced by PRDX4 depletion was accompanied by increased levels of reactive oxygen species (ROS), DNA damage and a DNA-PKcs-governed DNA damage response. As such, PRDX4 depletion also sensitized cells and tumors to ionizing radiation. The source of ROS that created a dependency on PRDX4 was attributed to NADPH oxidase 4 (NOX4), which localizes to the ER membrane. The functional requirement for PRDX4 was correlated with cellular NADPH levels across different models of PDAC and could be rescued by depletion of NOX4 or NADPH. As such, this study has identified NOX4 as a link between metabolic deregulation and ER-specific redox vulnerability in PDAC. Since PRDX4 is not an essential gene in normal tissues, our work also suggests that PRDX4 represents a novel therapeutic target for pancreatic cancer that may be particularly potent in combination with radiotherapy.

cancer biology↗